Fibroblast Growth Factor 2 Is Produced By Renal Tubular Cells to Act as a Paracrine Factor in Maladaptive Kidney Repair After Cisplatin Nephrotoxicity.

Kidney repair after injury involves the cross-talk of injured kidney tubules with interstitial fibroblasts and immune cells. Although tubular cells produce multiple cytokines, the role and regulation of specific cytokines in kidney repair are largely undefined. In this study, we detected the induction of fibroblast growth factor 2 (FGF2) in mouse kidneys after repeated low-dose cisplatin (RLDC) treatment and in RLDC-treated renal proximal tubule cells in vitro. We further detected FGF2 in the culture medium of RLDC-treated renal tubular cells but not in the medium of control cells, indicating that RLDC induces FGF2 expression and secretion. Compared with the medium of control cells, the medium of RLDC-treated renal tubular cells was twice as effective in promoting fibroblast proliferation. Remarkably, the proliferative effect of the RLDC-treated cell medium was diminished by FGF2-neutralizing antibodies. In addition, the RLDC-treated cell medium induced the expression of fibrosis-related proteins, which was partially suppressed by FGF2-neutralizing antibodies. In mice, FGF2 deficiency partially prevented RLDC-induced decline in kidney function, loss of kidney weight, renal fibrosis, and inflammation. Together, these results indicate that FGF2 is produced by renal tubular cells after kidney injury and acts as an important paracrine factor in maladaptive kidney repair and disease progression.

Tubular cell senescence promotes maladaptive kidney repair and chronic kidney disease after cisplatin nephrotoxicity.

Cisplatin is a widely used chemotherapy drug; however, it induces both acute and chronic kidney diseases (CKD) in patients with cancer. The pathogenesis of cisplatin-induced CKD is unclear, and effective renoprotective approaches are not available. Here, we report that repeated low-dose cisplatin (RLDC) treatment of C57BL/6 mice induced chronic cellular senescence in kidney tubules, accompanied with tubular degeneration and profibrotic phenotype transformation that culminated in maladaptive repair and renal fibrosis. Suppression of tubular senescence by senolytic drugs ABT-263 and Fisetin attenuated renal fibrosis and improved tubular repair, as indicated by restoration of tubular regeneration and renal function. In vitro, RLDC also induced senescence in mouse proximal tubular (BUMPT) cells. ABT-263 eliminated senescent BUMPT cells following RLDC treatment, reversed the profibrotic phenotype of the cells, and increased their clonogenic activity. Moreover, ABT-263 alleviated the paracrine effect of RLDC-treated BUMPT cells on fibroblasts for fibrosis. Consistently, knockdown of p16 suppressed post-RLDC senescence and fibrotic changes in BUMPT cells and alleviated their paracrine effects on renal fibroblast proliferation. These results indicate that persistent induction of tubular senescence plays an important role in promoting cisplatin-induced CKD. Targeting senescent tubular cells may be efficient for improvement of kidney repair and for the prevention and treatment of cisplatin-induced CKD.

The situation analysis of hot dry rock geothermal energy development in China-based on structural equation modeling.

In order to combat climate change, China proposed a dual carbon target in 2020. (China's 2030 and 2060 Goals). If the goals can be achieved as scheduled, it will help the world achieve the carbon neutrality goal earlier and improve the increasingly serious global climate. Reducing the utilization of fossil fuels and increasing the share of clean energy in primary energy are major ways to achieve China's 2030 and 2060 Goals. As a geothermal resource with large reserves, high energy storage, clean and pollution-free, dry hot rocks can effectively contribute to the goals. To promote the utilization of dry hot rock, this paper quantitatively studies the factors affecting the development and utilization of dry hot rock, and first summarizes five major influencing factors, namely, resources, environment, market, exploration and development technology, and effective use of technology. Then questionnaire survey based on these five factors was designed and distributed by email to geothermal energy experts in universities (China University of Geosciences, etc.) and research institutions (Chinese Academy of Sciences, etc.). Finally, the questionnaires are subjected to a reliability credibility test and validity analysis to remove the non-conforming items, and the structural equation model was constructed to analyze the data. The results show that environment, market and exploration and development technology have significant effects on dry hot rocks, while resources and effective use of technology have insignificant effects on dry hot rocks. Finally, some suggestions to promote the development of dry hot rocks in China are proposed.

p53 in Proximal Tubules Mediates Chronic Kidney Problems after Cisplatin Treatment.

Nephrotoxicity is a major side-effect of cisplatin in chemotherapy, which can occur acutely or progress into chronic kidney disease (CKD). The protein p53 plays an important role in acute kidney injury induced by cisplatin, but its involvement in CKD following cisplatin exposure is unclear. Here, we address this question by using experimental models of repeated low-dose cisplatin (RLDC) treatment. In mouse proximal tubular BUMPT cells, RLDC treatment induced p53 activation, apoptosis, and fibrotic changes, which were suppressed by pifithrin-α, a pharmacologic inhibitor of p53. In vivo, chronic kidney problems following RLDC treatment were ameliorated in proximal tubule-specific p53-knockout mice (PT-p53-KO mice). Compared with wild-type littermates, PT-p53-KO mice showed less renal damage (KIM-1 positive area: 0.97% vs. 2.5%), less tubular degeneration (LTL positive area: 15.97% vs. 10.54%), and increased proliferation (Ki67 positive area: 2.42% vs. 0.45%), resulting in better renal function after RLDC treatment. Together, these results indicate that p53 in proximal tubular cells contributes significantly to the development of chronic kidney problems following cisplatin chemotherapy.

Single-Nucleus Transcriptional Profiling of Chronic Kidney Disease after Cisplatin Nephrotoxicity.

Cisplatin induces both acute and chronic nephrotoxicity during chemotherapy in patients with cancer. Presented here is the first study of single-nucleus RNA sequencing (snRNA-seq) of cisplatin-induced nephrotoxicity. Repeated low-dose cisplatin treatment (RLDC) led to decreases in renal function and kidney weight in mice at 9 weeks. The kidneys of these mice showed tubular degeneration and dilation. snRNA-seq identified 16 cell types and 17 cell clusters in these kidneys. Cluster-by-cluster comparison demonstrated cell type-specific changes in gene expression and identified a unique proximal tubule (PT) injury/repair cluster that co-expressed the injury marker kidney injury molecule-1 (Kim1) and the proliferation marker Ki-67. Compared with control, post-RLDC kidneys had 424 differentially expressed genes in PT cells, including tubular transporters and cytochrome P450 enzymes involved in lipid metabolism. snRNA-seq also revealed transcriptional changes in potential PT injury markers (Krt222, Eda2r, Ltbp2, and Masp1) and repair marker (Bex4). RLDC induced inflammation and proinflammatory cytokines (RelB, TNF-α, Il7, Ccl2, and Cxcl2) and the expression of fibrosis markers (fibronectin, collagen I, connective tissue growth factor, vimentin, and α-smooth muscle actin). Together, these results provide new insights into RLDC-induced transcriptional changes at the single-cell level that may contribute to the development of chronic kidney problems in patients with cancer after cisplatin chemotherapy.

Research on the impact of COVID-19 on Chinese small and medium-sized enterprises: Evidence from Beijing.

COVID-19 leads small and medium-sized enterprises (SMEs) to survive very hard. The development difficulties of SMEs lead to weak employment and GDP growth in various countries. In the process of COVID-19's continuous spread, what is the major reason for the difficulties of SMEs? This paper hopes to answer this question by studying SMEs in Beijing. On this basis, this paper uses structural equation model (SEM) to study the relatively fast recovery of SMEs in Beijing, China, to explore the factors affecting SMEs in the pandemic. After detailed desk research and interviews with relevant entrepreneurs, this paper collects 234 valid questionnaires from SMEs in various industries in Beijing with the help of Federation of Industry and Commerce and Chamber of Commerce in Beijing. Then the data is analyzed with the SEM, which shows the relationship between cash flow from financing activities, markets, employees, costs, government policies and the impact of the pandemic. Finally, an impact model of the pandemic on SMEs is established. The result of the model indicates that the direct effect of the pandemic on the market is the most prominent, and government policies can significantly reduce the negative impact of the pandemic on SMEs indirectly. Based on this, this paper puts forward some policy suggestions, such as the targeted issuance of consumption vouchers and the reduction of administrative barriers. This will enable megacities in various countries to improve policy support for SMEs and promote the recovery and development of SMEs.

Autophagy in Cisplatin Nephrotoxicity during Cancer Therapy.

Cisplatin is a widely used chemotherapeutic agent but its clinical use is often limited by nephrotoxicity. Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged or dysfunctional cellular organelles for maintaining cell homeostasis. Upon cisplatin exposure, autophagy is rapidly activated in renal tubule cells to protect against acute cisplatin nephrotoxicity. Mechanistically, the protective effect is mainly related to the clearance of damaged mitochondria via mitophagy. The role and regulation of autophagy in chronic kidney problems after cisplatin treatment are currently unclear, despite the significance of research in this area. In cancers, autophagy may prevent tumorigenesis, but autophagy may reduce the efficacy of chemotherapy by protecting cancer cells. Future research should focus on developing drugs that enhance the anti-tumor effects of cisplatin while protecting kidneys during cisplatin chemotherapy.

Proximal Tubule p53 in Cold Storage/Transplantation-Associated Kidney Injury and Renal Graft Dysfunction.

Kidney injury associated with cold storage/transplantation is a primary factor for delayed graft function and poor outcome of renal transplants. p53 contributes to both ischemic and nephrotoxic kidney injury, but its involvement in kidney cold storage/transplantation is unclear. Here, we report that p53 in kidney proximal tubules plays a critical role in cold storage/transplantation kidney injury and inhibition of p53 can effectively improve the histology and function of transplanted kidneys. In a mouse kidney cold storage/transplantation model, we detected p53 accumulation in proximal tubules in a cold storage time-dependent manner, which correlated with tubular injury and cell death. Pifithrin-α, a pharmacologic p53 inhibitor, could reduce acute tubular injury, apoptosis and inflammation at 24 h after cold storage/transplantation. Similar effects were shown by the ablation of p53 from proximal tubule cells. Notably, pifithrin-α also ameliorated kidney injury and improved the function of transplanted kidneys in 6 days when it became the sole life-supporting kidney in recipient mice. in vitro, cold storage followed by rewarming induced cell death in cultured proximal tubule cells, which was accompanied by p53 activation and suppressed by pifithrin-α and dominant-negative p53. Together, these results support a pathogenic role of p53 in cold storage/transplantation kidney injury and demonstrate the therapeutic potential of p53 inhibitors.

A credit risk assessment model of borrowers in P2P lending based on BP neural network.

Peer-to-Peer (P2P) lending provides convenient and efficient financing channels for small and medium-sized enterprises and individuals, and therefore it has developed rapidly since entering the market. However, due to the imperfection of the credit system and the influence of cyberspace restrictions, P2P network lending faces frequent borrower credit risk crises during the transaction process, with a high proportion of borrowers default. This paper first analyzes the basic development of China's P2P online lending and the credit risks of borrowers in the industry. Then according to the characteristics of P2P network lending and previous studies, a credit risk assessment indicators system for borrowers in P2P lending is formulated with 29 indicators. Finally, on the basis of the credit risk assessment indicators system constructed in this paper, BP neural network is built based on the BP algorithm, which is trained by the LM algorithm (Levenberg-Marquardt), Scaled Conjugate Gradient, and Bayesian Regularization respectively, to complete the credit risk assessment model. By comparing the results of three mentioned training methodologies, the BP neural network trained by the LM algorithm is finally adopted to construct the credit risk assessment model of borrowers in P2P lending, in which the input layer node is 9, the hidden layer node is 11 and output layer node is 1. The model can provide practical guidance for China and other countries' P2P lending platforms, and therefore to establish and improve an accurate and effective borrower credit risk management system.

FOXO1-Mediated Downregulation of RAB27B Leads to Decreased Exosome Secretion in Diabetic Kidneys.

Exosomes have been implicated in diabetic kidney disease (DKD), but the regulation of exosomes in DKD is largely unknown. Here, we have verified the decrease of exosome secretion in DKD and unveiled the underlying mechanism. In Boston University mouse proximal tubule (BUMPT) cells, high-glucose (HG) treatment led to a significant decrease in exosome secretion, which was associated with specific downregulation of RAB27B, a key guanosine-5'-triphosphatase in exosome secretion. Overexpression of RAB27B restored exosome secretion in HG-treated cells, suggesting a role of RAB27B downregulation in the decrease of exosome secretion in DKD. To understand the mechanism of RAB27B downregulation, we conducted bioinformatics analysis that identified FOXO1 binding sites in the Rab27b gene promoter. Consistently, HG induced phosphorylation of FOXO1 in BUMPT cells, preventing FOXO1 accumulation and activation in the nucleus. Overexpression of nonphosphorylatable, constitutively active FOXO1 led to the upregulation of RAB27B and an increase in exosome secretion in HG-treated cells. In vivo, compared with normal mice, diabetic mice showed increased FOXO1 phosphorylation, decreased RAB27B expression, and reduced exosome secretion. Collectively, these results unveil the mechanism of exosome dysfunction in DKD where FOXO1 is phosphorylated and inactivated in DKD, resulting in RAB27B downregulation and the decrease of exosome secretion.

p53/microRNA-214/ULK1 axis impairs renal tubular autophagy in diabetic kidney disease.

Dysregulation of autophagy in diabetic kidney disease (DKD) has been reported, but the underlying mechanism and its pathogenic role remain elusive. We show that autophagy was inhibited in DKD models and in human diabetic kidneys. Ablation of autophagy-related gene 7 (Atg7) from kidney proximal tubules led to autophagy deficiency and worse renal hypertrophy, tubular damage, inflammation, fibrosis, and albuminuria in diabetic mice, indicating a protective role of autophagy in DKD. Autophagy impairment in DKD was associated with the downregulation of unc-51-like autophagy-activating kinase 1 (ULK1), which was mediated by the upregulation of microRNA-214 (miR-214) in diabetic kidney cells and tissues. Ablation of miR-214 from kidney proximal tubules prevented a decrease in ULK1 expression and autophagy impairment in diabetic kidneys, resulting in less renal hypertrophy and albuminuria. Furthermore, blockade of p53 attenuated miR-214 induction in DKD, leading to higher levels of ULK1 and autophagy, accompanied by an amelioration of DKD. Compared with nondiabetic samples, renal biopsies from patients with diabetes showed induction of p53 and miR-214, associated with downregulation of ULK1 and autophagy. We found a positive correlation between p53/miR-214 and renal fibrosis, but a negative correlation between ULK1/LC3 and renal fibrosis in patients with diabetes. Together, these results identify the p53/miR-214/ULK1 axis in autophagy impairment in diabetic kidneys, pinpointing possible therapeutic targets for DKD.

Decreased secretion and profibrotic activity of tubular exosomes in diabetic kidney disease.

Tubular changes contribute to the development of renal pathologies in diabetic kidney disease (DKD), including interstitial fibrosis. It is unclear how tubular cells relay signals to interstitial fibroblasts. Recently, exosomes have been recognized as crucial mediators of intercellular communication. We hypothesized that exosomes secreted from tubular cells may stimulate fibroblasts for interstitial fibrosis in DKD. In this study, we isolated and purified exosomes from the renal cortex of DKD mice and high glucose-treated mouse proximal tubular cells. Compared with nondiabetic mice, exosome secretion in kidney tissues decreased in DKD mice. Likewise, high glucose incubation reduced exosome secretion in mouse kidney proximal tubular BUMPT cells. To study the effect of tubular cell exosomes on fibroblasts, exosomes from BUMPT cells were added to renal fibroblast NRK-49F cell cultures. Notably, exosomes from high glucose conditioned BUMPT cells induced higher proliferation, significant morphological change, and substantial production of fibronectin, α-smooth muscle actin, and collagen type Ι in NRK-49F fibroblasts. Proteomics analysis was further performed to profile the proteins within tubular cell exosomes. Interestingly, 22 proteins were found to be differentially expressed between tubular exosomes derived from high glucose conditioned cells and those from normal glucose conditioned cells. Cytoscape analysis suggested the existence of two protein-protein interaction networks in these exosomal differentially expressed proteins. While one of the protein-protein interaction networks comprised enolase 1 (Eno1), heat shock protein family A member 8 (Hspa8), thioredoxin 1 (Txn1), peptidylprolyl isomerase A (Ppia), phosphoglycerate kinase 1 (Pgk1), DNA topoisomerase II-ß (Top2b), and ß-actin (Actb), the other had the family proteins of human leucocyte antigen F (Ywhag), a component of the ND10 nuclear body (Ywhae), interferon regulatory factor-8 (Ywhaq), and human leucocyte antigen A (Ywhaz). Gene expression analysis via Nephroseq showed a correlation of Eno1 expression with DKD clinical manifestation. In conclusion, DKD is associated with a decrease in exosome secretion in renal tubular cells. Exosomes from high glucose conditioned tubular cells may regulate the proliferation and activation of fibroblasts, contributing to the paracrine signaling mechanism responsible for the pathological onset of renal interstitial fibrosis in DKD.

miR-214 represses mitofusin-2 to promote renal tubular apoptosis in ischemic acute kidney injury.

Disruption of mitochondrial dynamics is an important pathogenic event in both acute and chronic kidney diseases, but the underlying mechanism remains poorly understood. Here, we report the regulation of mitofusin-2 (Mfn2; a key mitochondrial fusion protein) by microRNA-214 (miR-214) in renal ischemia-reperfusion that contributes to mitochondrial fragmentation, renal tubular cell death, and ischemic acute kidney injury (AKI). miR-214 was induced, whereas Mfn2 expression was decreased, in mouse ischemic AKI and cultured rat kidney proximal tubular cells (RPTCs) following ATP depletion treatment. Overexpression of miR-214 decreased Mfn2. Conversely, inhibition of miR-214 with anti-miR-214 prevented Mfn2 downregulation in RPTCs following ATP depletion. Anti-miR-214 further ameliorated mitochondrial fragmentation and apoptosis, whereas overexpression of miR-214 increased apoptosis, in ATP-depleted RPTCs. To test regulation in vivo, we established a mouse model with miR-214 specifically deleted from kidney proximal tubular cells (PT-miR-214-/-). Compared with wild-type mice, PT-miR-214-/- mice had less severe tissue damage, fewer apoptotic cells, and better renal function after ischemic AKI. miR-214 induction in ischemic AKI was suppressed in PT-miR-214-/- mice, accompanied by partial preservation of Mfn2 in kidneys. These results unveil the miR-214/Mfn2 axis that contributes to the disruption of mitochondrial dynamics and tubular cell death in ischemic AKI, offering new therapeutic targets.

P53 in kidney injury and repair: Mechanism and therapeutic potentials.

Acute kidney injury (AKI) is a major kidney disease with poor clinical outcome. Besides its acute consequence of high mortality, AKI may also contribute significantly to the occurrence and progression of chronic kidney diseases (CKD). Accumulating evidence has demonstrated that maladaptive and incomplete kidney repair after AKI leads to the development of renal fibrosis and, ultimately, CKD. p53, a well-known tumor suppressor, plays a critical role in AKI and subsequent kidney repair through the regulation of various cell biologic processes, including apoptosis, cell cycle arrest, and autophagy. Despite the notable progress in deciphering the involvement of p53 in kidney injury and repair, the underlying mechanisms of p53 in these pathological processes remain largely unknown. Further investigation in this area is essential for the application of p53 as therapeutic target to prevent and treat AKI or impede its progression to CKD. In this review, we summarize the recent advances in understanding p53 regulation of AKI and kidney repair, pinpoint the potential of p53 as a therapeutic target, and present future research interests and directions.

Dicer deficiency in proximal tubules exacerbates renal injury and tubulointerstitial fibrosis and upregulates Smad2/3.

Renal fibrosis is a common pathological feature in chronic kidney disease (CKD), including diabetic kidney disease (DKD) and obstructive nephropathy. Multiple microRNAs have been implicated in the pathogenesis of both DKD and obstructive nephropathy, although the overall role of microRNAs in tubular injury and renal fibrosis in CKD is unclear. Dicer (a key RNase III enzyme for microRNA biogenesis) was specifically ablated from kidney proximal tubules in mice via the Cre-lox system to deplete micoRNAs. Proximal tubular Dicer knockout (PT- Dicer KO) mice and wild-type (WT) littermates were subjected to streptozotocin (STZ) treatment to induce DKD or unilateral ureteral obstruction (UUO) to induce obstructive nephropathy. Renal hypertrophy, renal tubular apoptosis, kidney inflammation, and tubulointerstitial fibrosis were examined. Compared with WT mice, PT- Dicer KO mice showed more severe tubular injury and renal inflammation following STZ treatment. These mice also developed higher levels of tubolointerstitial fibrosis. Meanwhile, PT- Dicer KO mice had a significantly higher Smad2/3 expression in kidneys than WT mice (at 6 mo of age) in both control and STZ-treated mice. Similarly, UUO induced more severe renal injury, inflammation, and interstitial fibrosis in PT- Dicer KO mice than WT. Although we did not detect obvious Smad2/3 expression in sham-operated mice (2-3 mo old), significantly more Smad2/3 was induced in obstructed PT- Dicer KO kidneys. These results supported a protective role of Dicer-dependent microRNA synthesis in renal injury and fibrosis development in CKD, specifically in DKD and obstructive nephropathy. Depletion of Dicer and microRNAs may upregulate Smad2/3-related signaling pathway to enhance the progression of CKD.

DNA damage response in nephrotoxic and ischemic kidney injury.

DNA damage activates specific cell signaling cascades for DNA repair, cell cycle arrest, senescence, and/or cell death. Recent studies have demonstrated DNA damage response (DDR) in experimental models of acute kidney injury (AKI). In cisplatin-induced AKI or nephrotoxicity, the DDR pathway of ATR/Chk2/p53 is activated and contributes to renal tubular cell apoptosis. In ischemic AKI, DDR seems more complex and involves at least the ataxia telangiectasia mutated (ATM), a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and p53; however, while ATM may promote DNA repair, p53 may trigger cell death. Targeting DDR for kidney protection in AKI therefore relies on a thorough elucidation of the DDR pathways in various forms of AKI.

Finite element study of human pelvis model in side impact for Chinese adult occupants.

OBJECTIVE: The occupant's pelvis is very vulnerable to side collision in road accidents. Finite element (FE) studies on pelvic injury help to design occupant protection devices to improve vehicle safety. This study was aimed to develop a highly biofidelic pelvis model of Chinese adults and assess its sensitivity to variations in pelvis cortical bone thickness, bone material properties, and loading conditions. METHODS: In this study, 4 different FE models of the pelvis were developed from the computed tomography (CT) data of a volunteer representing the 50th percentile Chinese male. Two of them were meshed using entirely hexahedral elements with variable and constant cortical thickness distribution (the V-Hex and C-Hex models), and the others were modeled with hexahedral elements for cancellous bone and variable or constant thickness shell elements for cortical bone (the V-HS and C-HS models). In model developments, the semi-automatic multiblock meshing approach was employed to maintain the pelvis geometric curvature and generate a high-quality hexahedral mesh. Then, several simulations with postmortem human subjects (PMHS) tests were performed to obtain the most accurate model in predicting pelvic injury. Based on the most accurate model, sensitivity studies were conducted to analyze the effects of the cortex thickness, Young's modulus of the cortical and cancellous bone, impactor velocity, and impactor with or without padding on the biomechanical responses and injuries of pelvis. RESULTS: The results indicate that the models with variable cortical bone thickness can give more accurate predictions than those with constant cortical thickness. Both the V-Hex and V-HS models are favorable for simulating pelvic response and injury, but the simulation results of the V-Hex model agree with the tests better. The sensitivity study shows that pelvic response is more sensitive to alterations in the Young's modulus of cortical bone than cancellous bone. Compared to failure displacement, peak force is more sensitive to the cortical bone thickness. However, displacement is more sensitive to the Young's modulus of cancellous bone than peak force. The padding attached on the impactor plays a significant role in absorbing the impact energy and alleviating pelvic injury. CONCLUSIONS: The all-hex meshing method with variable cortical bone thickness has the highest accuracy but is time-consuming. The cortical bone plays a determining role in resisting pelvic fracture. Peak impact force appears to be a reasonable injury predictor for pelvic injury assessment. Some appropriate energy absorbers installed in the car door can significantly reduce pelvic injury and will be beneficial for occupant protection.

C5aR, TNF-α, and FGL2 contribute to coagulation and complement activation in virus-induced fulminant hepatitis.

BACKGROUND & AIMS: Viral fulminant hepatitis (FH) is a disease with a high mortality rate. Activation of the complement system correlates with the development of FH. However, the key factors mediating complement activation in FH remain elusive. METHODS: Liver tissues were isolated from FH patients infected by hepatitis B virus (HBV) and from mice infected with murine hepatitis virus strain 3 (MHV-3). Wild type mice were treated with or without antagonists of C5aR or TNF-α, and mice deficient for C5aR (C5aR(-/-)), Fgl2 (Fgl2(-/-)), and Tnfα (Tnfα(-/-)) mice were not treated with the antagonists. C5b-9, C5aR, FGL2, CD31, CD11b, fibrin, TNF-α, and complement C3 cleavage products were detected by immunohistochemistry, immunofluorescence, or ELISA. Sorted liver sinusoidal endothelial cells (LSECs) or myeloid-derived (CD11b(+)) cells were stimulated with C5a, TNF-α or MHV-3 in vitro. The mRNA expressions levels of Fgl2 and Tnfα were determined by qRT-PCR analyses. RESULTS: We observed that complement activation, coagulation and pro-inflammatory cytokine production were upregulated in the HBV(+) patients with FH. Similar observations were made in the murine FH models. Complement activation and coagulation were significantly reduced in MHV-3 infected mice in the absence of C5aR, Tnfα or Fgl2. The MHV-3 infected C5aR(-/-) mice exhibited reduced numbers of infiltrated inflammatory CD11b(+) cells and a reduced expression of TNF-α and FGL2. Moreover, C5a administration stimulated TNF-α production by CD11b(+) cells, which in turn promoted the expression of FGL2 in CD31(+) LSEC-like cells in vitro. Administration of antagonists against C5aR or TNF-α ameliorated MHV-3-induced FH. CONCLUSIONS: Our results demonstrate that C5aR, TNF-α, and FGL2 form an integral network that contributes to coagulation and complement activation, and suggest that those are potential therapeutic targets in viral FH intervention.

DNA damage response in renal ischemia-reperfusion and ATP-depletion injury of renal tubular cells.

Renal ischemia-reperfusion leads to acute kidney injury (AKI) that is characterized pathologically by tubular damage and cell death, followed by tubular repair, atrophy and interstitial fibrosis. Recent work suggested the possible presence of DNA damage response (DDR) in AKI. However, the evidence is sketchy and the role and regulation of DDR in ischemic AKI remain elusive. In this study, we demonstrated the induction of phosphorylation of ATM, H2AX, Chk2 and p53 during renal ischemia-reperfusion in mice, suggesting DDR in kidney tissues. DDR was also induced in vitro during the recovery or "reperfusion" of renal proximal tubular cells (RPTCs) after ATP depletion. DDR in RPTCs was abrogated by supplying glucose to maintain ATP via glycolysis, indicating that the DDR depends on ATP depletion. The DDR was also suppressed by the general caspase inhibitor z-VAD and the overexpression of Bcl-2, supporting a role of apoptosis-associated DNA damage in the DDR. N-acetylcysteine (NAC), an antioxidant, suppressed the phosphorylation of ATM and p53 and, to a less extent, Chk2, but NAC increased the phosphorylation and nuclear foci formation of H2AX. Interestingly, NAC increased apoptosis, which may account for the observed H2AX activation. Ku55933, an ATM inhibitor, blocked ATM phosphorylation and ameliorated the phosphorylation of Chk2 and p53, but it increased H2AX phosphorylation and nuclear foci formation. Ku55933 also increased apoptosis in RPTCs following ATP depletion. The results suggest that DDR occurs during renal ischemia-reperfusion in vivo and ATP-depletion injury in vitro. The DDR is partially induced by apoptosis and oxidative stress-related DNA damage. ATM, as a sensor in the DDR, may play a cytoprotective role against tubular cell injury and death.

hMSH2 recruits ATR to DNA damage sites for activation during DNA damage-induced apoptosis.

DNA damage response (DDR) activates a complex signaling network that triggers DNA repair, cell cycle arrest, and/or cell death. Depending on the type and severity of DNA lesion, DDR is controlled by "master" regulators including ATM and ATR protein kinases. Cisplatin, a major chemotherapy drug that cross-links DNA, induces ATR-dependent DDR, resulting in apoptosis. However, it is unclear how ATR is activated. To identify the key regulators of ATR, we analyzed the proteins that associate with ATR after cisplatin treatment by blue native-PAGE and co-immunoprecipitation. The mismatch repair protein hMSH2 was found to be a major ATR-binding protein. Functionally, ATR activation and its recruitment to nuclear foci during cisplatin treatment were attenuated, and DNA damage signaling, involving Chk2, p53, and PUMA-α, was suppressed in hMSH2-deficient cells. ATR activation induced by the DNA methylating agent N-methyl-N-nitrosourea was also shown to be hMSH2-dependent. Intriguingly, hMSH2-mediated ATR recruitment and activation appeared independent of replication protein A, Rad17, and the Rad9-Hus1-Rad1 protein complex. Together the results support a hMSH2-dependent pathway of ATR activation and downstream Chk2/p53 signaling.